NMR studies of the structure and dynamics of staphylococcal nuclease (SNase) were carried out in order to elucidate the catalytic function of this model enzyme. We studied (1) the molecular dynamics of wild type SNase and (2) the three dimensional (3D) structure of SNase mutants. (1) SNase dynamics. Heteronuclear 2D NMR pulse sequences were used to obtain accurate values of 15N and 13C labeled sites in SNase. The relaxation parameters of the 14 alanine, 22 leucine, and 4 methionine methyl carbons in SNase were measured and analyzed using the model-free approach. Little internal motion was found at the ` and ~ sites of the alanine residues. In contrast significant internal motion of nearly one half of the leucine sidechains was observed in SNase liganded to Ca2+ and pdTp. In the absence of the ligands, internal motions increased substantially. These results were interesting because all leucine sidechains are buried, suggesting a dynamic environment in portions of the protein interior. In addition, two of the methionine residues near the protein surface, showed substantially greater internal mobility than the most flexible leucine sidechains. (2) Mutant structure and function. The SNase mutant, G50F/V51N ~SNase, is considerably more stable than the wild type enzyme, but is ca. 100 fold less active. We have determined the 3D structure in solution to high resolution based on over 2000 NOE constraints. The structures of the mutant and wild type proteins are essentially identical except for a few residues near the active site. The difference in stability is due to the replacement of a disordered loop of the wild type enzyme with a well structured tight turn in the mutant. The lower activity of the mutant protein appears to result from the movement of the active site Glu-43 sidechain from its position in the wild type structure. The significance of the project lies in the information about (1) the range and time scales of protein structural fluctuations provided by dynamics studies and (2) the relationship between structure and function that comes from comparing the structures of wild type and mutant proteins. In addition, many novel NMR experiments have been developed using SNase as a model system.